Cigarette smoking is the single largest preventable source of morbidity and mortality in North America and Europe. Twin and adoption studies indicate that a majority of risk for nicotine dependence (ND) is genetic. A genome-wide association (WGA) study of cigarettes per day (CPD), as a quantitative trait, was conducted with ~ 7500 individuals of European origin. Genotyping was done with Affymetrix 500K chips. A SNP in the alpha 3 nicotinic receptor subunit gene (CHRNA3) was associated with CPD as a quantitative trait (p = 0.00007). Analyses of CPD in a case-control mode (cases defined as smoking > 25 CPD versus controls < 5 CPD), was done in a second European-origin population of ~ 6200 controls and ~ 1740 cases, genotyped at ~ 6000 SNPs. In this second population, a CHRNA3 SNP was strongly associated with CPD (p = 0.0000026, odds ratio 1.30, 95% CI 1.17-1.48). These results are similar to a case-control study of ND by Saccone et al (2007), who reported association of several CHRNA3 SNPs (0.0003< p < 0.01). All the identified risk alleles (N = 7) at these SNPs lie on a single common haplotype, with ~ 38% allele frequency in persons of Euorpean origin. Because of strong linkage disequilibrium across the CHRNA3 and the adjacent CHRNA5 gene, additional genotyping is unlikely to identify causal variations. This proposal describes functional studies of CHRNA3 and CHRNA5 genes. Re-sequencing of CHRNA3 and CHRNA5 in ~ 200 ND persons will be employed to identify rare functional variants which might predispose to ND. These rare functional variants will be examined for linkage disequilibrium in a population of ~ 1500 ND individuals and ~ 1500 controls, all of European origin, to determine whether they might predispose to ND. CHRNA3 and CHRNA5 mRNA and protein will be assessed in post mortem human brain from individuals with the putative risk and protective haplotypes. CHRNA3 and CHRNA5 promoters sequences, from the risk and protective haplotypes, will be assessed in tissue culture for transcriptional efficiency. A mis-sense coding SNP in CHRNA5, which lies on the risk haplotype, will be assessed for change in binding affinties and ionic conductance. These results should identify the functional effects of promoter and transcribed SNPs in CHRNA3 and CHRNA5 genes. This study should identify the consequences of CHRNA3 and/or CHRNA5 gene sequences which increase risk for ND and should facilitate new drug development for treatment of ND.